The present invention relates to methods for the purification and separation of species, particularly those of biological interest, from free solutions wherein the methods employ soluble, small molecular weight ligands that are capable of specifically interacting with the species of interest.
The success of many research projects in the biotechnology arts is absolutely dependent upon the availability of techniques that allow for the separation and purification of one or more molecules of interest from complex mixtures comprising those molecules of interest. In this regard, several techniques are currently available for separating and purifying molecules of biological interest, such as proteins, from complex mixtures thereof. These techniques include, for example, affinity chromatography, ion exchange chromatography (IEC), size exclusion chromatography (SEC), high performance liquid chromatography (HPLC), hydrophobic interaction chromatography (HIC), different variations of membrane filtration such as ultrafiltration, microfiltration, reverse osmosis, and the like. However, while these separation techniques have proven useful in a variety of applications, there are often many more applications for which their use is limited.
In an effort to enhance the separation capabilities of these techniques, researchers have developed a number of similar molecular separation techniques, but which are based at least in part upon the ability of an affinity molecule to bind to a entity of interest, thereby rendering that entity of interest separable from the other components of a fluid mixture. One such affinity molecule-based separation technique is affinity chromatography, which separates biological molecules based upon their ability to specifically and selectively bind to an affinity matrix or gel. Affinity gels typically consist of a ligand-binding moiety immobilized on a gel support. For example, GB 2,178,742 utilized affinity chromatography to purify hemoglobin and its chemically modified derivatives based upon the fact that native (oxy)hemoglobin binds specifically to polyanionic functionalities of certain affinity gels. In this process, unmodified hemoglobin is retained by the affinity gel while modified hemoglobin, which cannot bind to the gel because its polyanion binding site is covalently occupied by a modifying agent, is eluted.
However, while affinity chromatography has proven useful for a variety of applications, there are inherent limitations to the technique. For example, because affinity chromatography is dependent upon the attachment of a ligand to a solid phase matrix (such as a polymer bead or other type of polymeric matrix), there exist significant conformational constraints on the attached ligand molecule as well as on the protein which becomes bound to the matrix-attached ligand molecule. Moreover, the solid phase matrix provides a potential site for non-specific binding of various components of a reaction mixture, thereby often resulting in less than completely efficient separations. Also, the overall capacity of an affinity chromatography system is limited by the available surface area of the solid phase and by the density to which that surface area may be substituted by ligand.
Another well known affinity molecule-based separation technique is affinity ultrafiltration, a technique that combines affinity binding with membrane-based ultrafiltration. More specifically, affinity ultrafiltration employs a large, polymeric affinity ligand to which a protein of interest is able to bind followed by ultrafiltration-based separation of the complexed polymeric affinity ligand from the remaining components of a mixture. (See, e.g., Mattiasson et al., Journal of Chromatography 283:323-330 (1984), Luong et al., Biotechnology and Bioengineering 31:516-520 (1988) and Male et al., Biotechnology and Bioengineering 35:87-93 (1990)). However, because large, polymeric affinity ligands are employed as the affinity binding molecule, significant limitations exist as to the separation capacity due to the inherent insolubility of such affinity ligands. Moreover, polymeric affinity ligands are often not readily available and provide relatively low yields and/or purification factors.
van Eijndhoven et al., Biotechnology and Bioengineering 48:406-414 (1995) have demonstrated that alterations in the ionic strength of a protein-containing solution may function to alter the apparent size of a protein in that solution. More specifically, van Eijndhoven et al. have demonstrated that the interaction of monovalent ions (such as Clxe2x88x92) with a protein such as bovine serum albumin may function to alter the hydrodynamic volume of the protein. However, it is currently unknown whether the binding of small monovalent ions to proteins provides a significant enough of a change in the hydrodynamic volume of the protein to render it separable from other similarly sized proteins in a complex mixture thereof using known separation techniques.
There is, therefore, a need to develop novel methods for separating and purifying species of interest from complex free solution mixtures which are not subject to the limitations inherent in other known affinity molecule-based separations. Specifically, there is a need for affinity molecule-based techniques which are not subject to the conformational constraints and non-specific binding problems associated with affinity chromatography. Moreover, there is also a need for novel affinity molecule-based techniques which are not subject to the above described limitations of the affinity ultrafiltration process. The present invention provides a solution to many of these problems.
The present invention is directed to novel methods for separating a species of interest from other different species present in a free solution mixture thereof which takes advantage of affinity and other interactions that occur between soluble, small molecular weight ligands and a species of interest. Specifically, the present invention is directed to a method for separating a first species of interest from a second species of interest in a free solution mixture comprising the first and second species of interest, where the first and second species of interest are different. The herein described method comprises the step of contacting the free solution mixture with a small molecular weight soluble ligand that interacts in free solution with the first species of interest but not to a significant extent with the second species of interest, thereby forming a first species of interest/ligand complex which is capable of being separated from the second species of interest by a variety of different molecular separation techniques including, for example, size exclusion chromatography and membrane filtration. The small molecular weight ligand has a smaller molecular weight than the first species of interest with which it interacts.
In particular embodiments, the first species of interest may be, for example, a protein or polypeptide (naturally or recombinantly produced), an amino acid, a colloid, an endotoxin, a virus, a nucleic acid, and the like. Small molecular weight ligands which find use in the present invention are soluble in free solution systems and include, for example, proteins, peptides, antibodies, nucleic acids, amino acids, organic and inorganic molecules, multi-ionic molecules, and the like. Typically, the interaction between the first species of interest and the ligand occurs through affinity, hydrophobic and/or ionic interactions.
In various embodiments of the present invention, the first and second species of interest may initially have about the same hydrodynamic volumes, molecular weights and/or isoelectric points, thereby rendering them difficult to separate with conventional separation techniques. One or more of these properties, however, may become different upon interaction by the ligand with the first species of interest. In cases where the hydrodynamic volume of the first species of interest/ligand complex and the second species of interest is about the same, one may often alter the pH or the ionic strength of the system to effectively increase the difference in the hydrodynamic volume between the first species of interest/ligand complex and the second species of interest, thereby enhancing the ability to separate them using conventional separation techniques.
In yet other embodiments of the present invention, one may dissociate and separate the first species of interest and ligand components of the complex which is initially separated from the second species of interest, thereby providing a relatively pure preparation of the first species of interest. Methods for inducing this dissociation generally include altering the pH of the system, altering one or more soluble components in the system, and the like.
Still other embodiments of the present invention will become apparent to the ordinarily skilled artisan upon a reading of the present specification.